Summary: During graft-versus-host disease (GVHD), donor T cells require Ezh2, which is a histone methyltransferase that primarily acts as an epigenetic regulator, for producing and sustaining effector T cells mediating host tissue injury (Blood 2013, Blood 2017). Using adoptive T cell therapy mouse models, we most recently demonstrate that Ezh2 serves as a key molecular gatekeeper for the generation of CD8 memory T cell precursors that can continually produce effector T cells in response to persisting antigen (Nat Commun 2017). However, our investigation of developing novel approaches to selectively target alloreactive effector T cells has been limited by the lack of understanding of why Ezh2 loss causes cell death of antigen-activated T cells. Stromal interaction molecule (Stim) proteins, Stim1 and Stim2, function as crucial dynamic endoplasmic reticulum (ER) Ca2+ sensors and modulators of Ca2+ signals in T cells. Upon T cell receptor (TCR) ligation, Stim1 activation leads to their translocation towards the plasma membrane, where they activate the Ca2+ channel Orai1, facilitating Ca2+ entry and initiating T cell responses. Conditional deletion of Stim1 leads to inhibition of GVHD in mice due to impaired effector differentiation. Surprisingly, we now find that Stim1 deletion rescues antigen-activated Ezh2-null T cells, leading to restored production of alloreactive effector T cells in mice and severe GVHD. Building upon these preliminary observations, we hypothesize that: A) Ezh2 and Stim1 operate coordinately to regulate the viability and function of antigen-driven T cells; and B) these Ezh2/Stim1-regulated molecular pathway(s) may be crucial for controlling alloreactive T cell-mediated GVHD. Our subsequent studies reveal mitochondrial Ca2+ (mCa2+) overload as the critical Stim1-dependent effector of Ezh2-mediated cell death. We further show that conditional deletion of the mitochondrial calcium uniporter (MCU), required for mCa2+ entry, leads to rescue of antigen-activated Ezh2-null T cells. Using conditional MCU-deficient T cells, we performed preliminary screens of natural compounds and, discovered that MCU activation in T cells using kaempferol, which is a flavonoid compound derived from plant-foods, leads to mCa2+overload and selective decrease of survival and expansion of activated wild-type but not MCU-null T cells. Our preliminary experiments showed that kaempferol treatment reduced GVHD in BALB/C mice receiving allogeneic C57BL/6 T cells. Altogether, these findings suggest that Ezh2 regulates the survival of antigen-specific effector T cells through modulation of cytosolic Ca2+ (cCa2+) entry, thereby limiting the amount of Ca2+ available for loading into mitochondria and protecting against cell death. Thus, targeting MCU by increasing mCa2+overload may represent an effective strategy to modulate GVHD. This is conceptually different from exiting prophylactic regimen using the calcineurin inhibitors (e.g. cyclosporine A and tacrolimus) that block Ca2+-induced NFAT activation. In this application, we propose three specific aims to test these hypotheses. In Aim-1, we will generate further mechanistic insight into how driven of Ezh2-/- effector regulated will autoimmune for Stim1-deletion rescues antigen- Ezh2-null T cells. Aim-2 will determine the role of MCU-regulated uptake in mediating cell death T cells the beneficial ffects of pharmacologically targeting MCU- m Ca 2+ loading in T cells on GVH and GVL responses in mice. Completion of these experiments provide novel insights into T cell biology, T cell-mediated inflammatory disorders such as GVHD and diseases, and lead to development of novel methods for improving the efficacy of immunotherapy chronic infections and cancer. mCa2+ . Finally, Aim-3 will examine e If reducing the toxicity of alloreactive T cells by pharmacologically activating MCU can achieve the goal of GVHD prevention while preserving GVL, a substantial improvement in allo-HSCT outcomes can be realized. Furthermore, the proposed studies may also have broad impact on better understanding of how tumor cells escape T cell immunity via the mechanisms associated with decreases of Ezh2 activity and mCa2+ loading in T cells. 1